Many polypeptide growth factors mediate their biological responses by binding to and activating cell surface receptors with intrinsic tyrosine kinase activity. Upon ligand binding, these receptors become autophosphorylated on multiple tyrosine residues and subsequently associate with intracellular molecules important for signal transduction (Ullrich, 1990). However, little is known about the structural determinants of the specificity of ligand-receptor binding and how this interaction results in receptor activation and in the transduction of pleiotropic biological effects.
Recent work on the Trk family of tyrosine kinase receptors has established that these molecules constitute signal-transducing receptors for a family of structurally and functionally related neurotrophic factors collectively known as the neurotrophins. Most of our knowledge on neurotrophic factors comes from work on nerve growth factor (NGF), a 118 amino acid polypeptide that controls the maturation and survival of sympathetic neurons, as well as subpopulations of sensory and central neurons (Levi-Montalcini, 1968; Thoenen, 1980; Thoenen, 1987.) Other neurotrophins include brain-derived neurotrophic factor (BDNF) (Barde 1982; Leibrock, 1989); neurotrophin-3 (NT-3); (Hohn, 1990; Maisonpierre, 1990; Ernfors, 1990; Rosenthal, 1990); and neurotrophin-4 (NT-4); (Hallbook, 1991; Ip, 1992) also named neurotrophin-5 (NT-5); (Berkemeier, 1991). The ability of neurotrophins to promote survival of peripheral and central neurons during development and after neuronal damage has stimulated the interest in these molecules as potential therapeutic agents for the treatment of neurodegenerative diseases and nervous system injuries.
The neurotrophins show about 50% sequence identities and display both overlapping and specific sets of neurotrophic activities on peripheral and central neurons. For example, all neurotrophins, in various proportions, support the survival of neural crest-derived sensory neurons [Thoenen, 1980; Lindsay, 1985; Hohn, 1990; Ip, 1992]. In contrast, survival of embryonic sympathetic neurons is supported only by NGF, while placode-derived sensory neurons are supported by BDNF and NT-3, but not by NGF [Thoenen, 1980; Lindsay, 1985; Hohn, 1990]. This specificity is believed to be achieved in part by the selective interaction between the different neurotrophins and the members of the Trk family of tyrosine kinase receptors expressed on the surface of distinct neuronal populations. Thus, whereas NGF binds only to p140.sup.trk (herein called TrkA) [Kaplan, 1991; Kaplan 1991a, Klein, 1991] BDNF and NT-4 interact exclusively with p145.sup.trkB (herein called TrkB) [Soppet, 1991; Squinto, 1991; Klein, 1991b; Ip, 1992], while NT-3 interacts with p145.sup.trkC (herein called TrkC) and to a lesser extent, also with TrkA and TrkB. [Cordon-Cardo, 1991; Lambelle, 1991; Klein, 1991b; Squinto, 1991].
Unlike Trk receptors, the low-affinity nerve growth factor receptor p75.sup.NGFR (herein called LNGFR) [Radeke, 1987; Johnson, 1986] recognizes each of the neurotrophins with a similar affinity [Rodriquez-Tebar, 1990; Ernfors, 1990; Hallbook, 1991; Rodriquez-Tebar, 1992]. Although LNGFR was initially postulated as a component of functional high-affinity NGF receptors (Hempstead, 1989; Hempstead, 1989] involved in mediating biological activity [Hempstead, 1989; Yan, 1991], emerging evidence disputes the direct role of this molecule in signal transduction [Westcamp, 1991, Glass, 1991; Ibaeez, 1992; Jing, 1992].
Information on neurotrophin-receptor interactions has been obtained from studies of structure-function relationships and the design of neurotrophin mutants with altered properties. For example, in copending parent application Ser. No. 07/847,369 now U.S. Pat. No. 5,349,055 filed on Mar. 6, 1992 entitled NEUROTROPHIC FACTORS HAVING ALTERED RECEPTOR BINDING SPECIFICITIES, which is incorporated by reference in its entirety herein, this approach was used to show that a positively charged interface in the NGF protein formed by Lys-32, Lys-34 and Lys-95 mediates the binding of NGF to LNGFR. NGF molecules mutated in these positions do not bind to LNGFR but retain binding to TrkA receptors and biological activity in cultures of primary neurons, thus demonstrating that TrkA alone is sufficient to mediate the biological activity of NGF in neuronal cells.
Most of the sequence variations among the neurotrophins occur in distinct regions, and initial studies using chimeric molecules between NGF and BDNF have shown that specific combinations of some of these variable sequences allow a broader spectrum of neurotrophic activities than those of the two wild type proteins (Ibanez, 1991; Surer, 1992). However, these studies did not identify specific receptor binding sites in the neurotrophins responsible for the observed differences in biological specificities.
The recent elucidation of the crystal structure of NGF has localized three quarters of the variable residues in three .beta.-hairpin loops and a reverse turn (McDonald, 1991). Other variable regions include part of a .beta.-strand and the amino (NH.sub.2) and carboxy (COOH) termini. The fact that most of the conserved amino acid residues in the neurotrophins play structural roles suggests that all four neurotrophins have very similar conformations, with individual differences restricted to the variable regions (McDonald, 1991). Surface loop regions are in general thought to be important for receptor binding of growth factors (Sclunegger, 1992; Daopin, 1992; Oefner, 1992). For example, in U.S. Pat. No. 5,134,121 issued on Jul. 28, 1992, agonists and antagonists of nerve growth factor are described which consist of peptides derived from the region between amino acid 26 and amino acid 40 of NGF.